The hallmark of progressive chronic kidney disease is the deposition of extracellular matrix proteins and tubulointerstitial fibrosis. Integrins mediate cell-extracellular matrix interaction and may play a role tubular epithelial injury. Murine primary tubular epithelial cells (TECs) express α₅-integrin, a fibroblast marker and the natural receptor for fibronectin. Microscopy localized α₅-integrin on E-cadherin-positive cells, confirming epithelial expression. The expression of α₅-integrin increased in TECs grown on fibronectin and occurred in parallel with an upregulation of α-smooth muscle actin (αSMA), a marker of epithelial-mesenchymal transition (EMT). Exposure of TECs to transforming growth factor (TGF)-β also increased TEC α₅-integrin expression in association with αSMA and EMT. Knock-down of α5-integrin expression with short interfering RNA attenuated the TGF-β induction of αSMA but did not alter morphologic EMT. Rather, α5-integrin was necessary for epithelial cell migration on fibronectin but not type IV collagen during cell spreading and epithelial wound healing in vitro. Immunohistochemistry revealed basolateral tubular epithelial α₅-integrin expression in mouse kidneys after unilateral ureteric obstruction but not in contralateral control kidneys. In patient biopsies of nondiabetic kidney disease, α₅-integrin expression was increased significantly in the renal interstitium. Focal basolateral staining was also detected in injured, but not in normal, tubular epithelium. In summary, these data show that TECs are induced to express α₅-integrin during EMT and tubular epithelial injury in vitro and in vivo. These results increase our understanding of the biology of integrins during EMT and tubular injury in chronic kidney disease.